Glyphosate (N-phosphonomethyl-glycine) is the active compound in Round Up, the most commonly used herbicide, it is used in agriculture, horticulture, and silviculture. Typically it is sprayed and absorbed through the leaves.
Glyphosate acts broadly against plant species and works via inhibition of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) which is a key enzyme in the shikimate pathway, essential for the production of aromatic amino acids. Glyphosate has a chemical structure similar to phosphoenolpyruvate (PEP), the natural EPSPS enzyme substrate, and thus competes with PEP for the enzyme active site. Inhibition of EPSPS disrupts amino acid synthesis and thereby kills the affected plant cells. Glyphosate is non-selective and it kills both weeds and crop plants.
The popularity of glyphosate as a herbicide is partly due to its low toxicity to animals. The shikimate pathway is only found in plants and bacteria. Monsanto originally patented glyphosate in the 1970s.
The observation that certain bacterial types were able to survive in glyphosate led to the discovery that some bacterial EPSPS enzymes are insensitive to glyphosate. This led to the development of Monsanto's transgenic Round Up Ready crops which express these glyphosate resistant bacterial EPSPS enzymes.
Current Roundup Ready crops include soybean, maize (corn), sorghum, canola, alfalfa, cotton and sugar beet. These crops greatly improved farmers' ability to control weeds, since glyphosate can be sprayed on fields without severely affecting the crops. As of 2005, 87% of USA soybean fields were planted with glyphosate-resistant crops (National Agriculture Statistics Service (2005) in Acreage eds. Johanns, M. & Wyatt, S. D. 6 30, (U.S. Dept, of Agriculture, Washington, D.C.)).
It is however found that Roundup Ready soybean crops, compared with the top conventional varieties, have a 6.7% lower yield (Charles Benbrook. Evidence of the Magnitude and Consequences of the Roundup Ready Soybean Yield Drag from University-Based Varietal Trials in 1998. Ag BioTech InfoNet Technical Paper Number 1). Conferring glyphosate resistance to a plant may involve significant fitness cost of said plant. Such fitness cost may, next to a decreased crop yield, also be expressed by decreased biomass accumulation over time.
An important mutation that has been found to give plants glyphosate resistance under field conditions is a single nucleotide change, altering the proline at position 106 in the EPSPS enzyme into a leucine (P106L). Many weed species around the world have independently developed this mutation but thus far other spontaneous mutations in EPSPS have not been found (Beerson of al. Plant Physiol, July 2002, Vol. 129, pp, 1265-1275 2002). Gassert et al. (J. Biol. Chem Vol 263(9) pp 4280-4289) describe structure, expression, and evolution of the 5-Enolpyruvylshikimate-3-phosphate Synthase Genes of Petunia and Tomato.
There is a need to identify further mutations that are useful in providing EPSPS enzymes that provide (improved) resistance to glyphosate, and plants carrying such mutation(s) and/or expressing such enzymes, that are thereby able to grow in the presence of (increased levels of) glyphosate. Such plants may be able to grow in the presence of higher concentrations of glyphosate in comparison with prior art plants, including glyphosate resistant plants, or show enhanced growth in the presence of similar concentrations of glyphosate in comparison with prior art plants, including glyphosate resistant plants.
Furthermore, there is also a need to identify mutations that further enhance glyphosate resistance of prior art glyphosate resistant plants or mutations that preferably reduce the fitness cost associated with the glyphosate resistance of said prior art glyphosate resistant plants.
It is an object of the present invention to provide for at least one of the above-mentioned needs.